When I did my plasmid in sequencing center, I choose the universal primer for the sequecing. However, I would like to know about the ineranl primers and when we need internal primer for sequecning and how can I check that? Also I want to knwo the ineranl primer.
Thank You.
Internal primers are used in 3 ways
1 after inserting a sequence into the plasmid a pcr of plasmid to internal sequence primer proves that the insert is incorporated into the plasmid and not just amplification of the target dna in the reagent mix
2 If your insert is long then sequencing primers can only sequence 800-1000 bases so to get sequence for a 3kb fragment you sequence from an internal primer at ,for example 700 bases to get the sequence of 750-1600 and again from a primer at 1550 to get sequence 1600-2400 etc. I have not heard of an inerani primer
In nested pcr a re amplification of diluted pcr product uses internal primers to clean up a dirty pcr product because the nested primers will often not exist in the unwanted amplimers
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